U.S. patent number 7,943,313 [Application Number 11/935,695] was granted by the patent office on 2011-05-17 for probe, probe set, probe-immobilized carrier, and genetic testing method.
This patent grant is currently assigned to Canon Kabushiki Kaisha. Invention is credited to Toshifumi Fukui, Hideto Kuribayashi, Hiroto Yoshii.
United States Patent |
7,943,313 |
Fukui , et al. |
May 17, 2011 |
Probe, probe set, probe-immobilized carrier, and genetic testing
method
Abstract
A nucleic acid probe for classification of pathogenic bacterial
species is capable of collectively detecting bacterial strains of
the same species and differentially detecting them from other
bacterial species. Any one of the base sequences of SEQ ID NOS. 70
to 72 and complementary or modified sequences thereof or a
combination of at least two of them is used for detecting the gene
of an infectious disease pathogenic bacterium.
Inventors: |
Fukui; Toshifumi (Yokohama,
JP), Yoshii; Hiroto (Tokyo, JP),
Kuribayashi; Hideto (Kawasaki, JP) |
Assignee: |
Canon Kabushiki Kaisha (Tokyo,
JP)
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Family
ID: |
39106341 |
Appl.
No.: |
11/935,695 |
Filed: |
November 6, 2007 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20080113363 A1 |
May 15, 2008 |
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Foreign Application Priority Data
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Nov 10, 2006 [JP] |
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2006-306007 |
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Current U.S.
Class: |
435/6.15;
435/91.2; 536/22.1; 536/24.3 |
Current CPC
Class: |
C12Q
1/689 (20130101); Y10T 436/145555 (20150115); B01J
2219/00608 (20130101) |
Current International
Class: |
C12Q
1/68 (20060101); C12P 19/34 (20060101); C07H
21/02 (20060101); C07H 21/04 (20060101) |
Field of
Search: |
;435/6,91.2
;536/22.1,24.3 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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8-89254 |
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Apr 1996 |
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JP |
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11-127899 |
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May 1999 |
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JP |
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2004-313181 |
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Nov 2004 |
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JP |
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2006-129828 |
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May 2006 |
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JP |
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2004-033720 |
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Apr 2004 |
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WO |
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Other References
Nadkarni et al. J. of Cllin. Microbiology, 2004, vol. 42(1), p.
5238-5244. McGarvey et al. Applied and Environmental Microbiology,
2004, vol. 70(7), p. 4267-4275. Lowe et al. Nucleic Acid Research,
1990, vol. 18(7), p. 1757-1761. cited by examiner .
The nucleic acid sequence search reports for SEQ ID No. 1, 22, 20
25, 70, 71, and 72. cited by examiner .
U.S. Appl. No. 11/996,744, filed Aug. 4, 206, Yoshii. cited by
other .
U.S. Appl. No. 11/935,914, filed Nov. 6, 2007, Kuribayashi,, et al.
cited by other .
U.S. Appl. No. 11/935,930, filed Nov. 6, 2007, Yshii, et al. cited
by other .
U.S. Appl. No. 11/935,807, filed Nov. 6, 2007, Kuribayashi, et al.
cited by other .
U.S. Appl. No. 11/935,789, filed Nov. 6, 2007, Fukui, et al. cited
by other .
U.S. Appl. No. 11/935,746, filed Nov. 6, 2007, Yoshii, et al. cited
by other .
U.S. Appl. No. 11/935,820, filed Nov. 6, 2007, Kuribayashi, et al.
cited by other .
U.S. Appl. No. 11/935,849, filed Nov. 6, 2007, Yoshii, et al. cited
by other .
European Search Report dated Apr. 11, 2008 in European Application
No. 07021829.2. cited by other.
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Primary Examiner: Horlick; Kenneth R.
Assistant Examiner: Tung; Joyce
Attorney, Agent or Firm: Fitzpatrick, Cella, Harper &
Scinto
Claims
What is claimed is:
1. An isolated and purified probe set for specifically detecting a
gene of infectious disease pathogenic bacterium, Prevotella
denticola, comprising first to third probes consisting of the
following base sequences (1) to (3) respectively: (1)
TCGATGACGGCATCAGATTCGAAGCA (SEQ ID NO. 70) or the fully
complementary sequence thereof; (2) AATGTAGGCGCCCAACGTCTGACT (SEQ
ID NO. 71) or the fully complementary sequence thereof; and (3)
ATGTTGAGGTCCTTCGGGACTCCT (SEQ ID NO. 72) or the fully complementary
sequence thereof.
2. A probe-immobilized carrier comprising a probe set according to
claim 1 arranged on a solid-phase carrier.
3. A probe-immobilized carrier according to claim 2, wherein the
probe-immobilized carrier comprises a fourth probe having any one
of the base sequences of SEQ ID NOS. 35 to 94 immobilized at a
position spaced from the first to third probes.
4. A kit for detecting a gene of Prevotella denticola, comprising:
a probe set according to claim 1; and a reagent for detecting a
reaction between the probe set and a target nucleic acid.
5. A kit according to claim 4, wherein the reagent contains a
primer for amplifying the gene of Prevotella denticola, and the
primer includes: an oligonucleotide having a base sequence of 5'
gctacaggcttaacacatgcaag 3' (SEQ ID NO: 20); and an oligonucleotide
having a base sequence of 5' atccagccgcaccttccggtac 3' (SEQ ID NO:
25).
6. A gene detection kit, comprising: a probe-immobilized carrier
according to claim 3; and a reagent for detecting a reaction
between a target nucleic acid and any one of the first to fourth
probes, wherein the reagent contains a primer including at least
one oligonucleotide selected from the following items (1) to (21)
and at least one oligonucleotide selected from the following items
(22) to (28): (1) an oligonucleotide having a base sequence of 5'
gcggcgtgcctaatacatgcaag 3' (SEQ ID NO: 1); (2) an oligonucleotide
having a base sequence of 5' gcggcaggcctaacacatgcaag 3' (SEQ ID NO:
2); (3) an oligonucleotide having a base sequence of 5'
gcggcaggcttaacacatgcaag 3' (SEQ ID NO: 3); (4) an oligonucleotide
having a base sequence of 5' gcggtaggcctaacacatgcaag 3' (SEQ ID NO:
4); (5) an oligonucleotide having a base sequence of 5'
gcggcgtgcttaacacatgcaag 3' (SEQ ID NO: 5); (6) an oligonucleotide
having a base sequence of 5' gcgggatgccttacacatgcaag 3' (SEQ ID NO:
6); (7) an oligonucleotide having a base sequence of 5'
gcggcatgccttacacatgcaag 3' (SEQ ID NO: 7); (8) an oligonucleotide
having a base sequence of 5' gcggcatgcttaacacatgcaag 3' (SEQ ID NO:
8); (9) an oligonucleotide having a base sequence of 5'
gcggcgtgcttaatacatgcaag 3' (SEQ ID NO: 9); 10) an oligonucleotide
having a base sequence of 5' gcggcaggcctaatacatgcaag 3' (SEQ ID NO:
10); (11) an oligonucleotide having a base sequence of 5'
gcgggatgctttacacatgcaag 3' (SEQ ID NO: 11); (12) an oligonucleotide
having a base sequence of 5' gcggcgtgcctaacacatgcaag 3' (SEQ ID NO:
12); (13) an oligonucleotide having a base sequence of 5'
gcggcgtgcataacacatgcaag 3' (SEQ ID NO: 13); (14) an oligonucleotide
having a base sequence of 5' gcggcatgcctaacacatgcaag 3' (SEQ ID NO:
14); (15) an oligonucleotide having a base sequence of 5'
gcggcgcgcctaacacatgcaag 3' (SEQ ID NO: 15); (16) an oligonucleotide
having a base sequence of 5' gcggcgcgcttaacacatgcaag 3' (SEQ ID NO:
16); (17) an oligonucleotide having a base sequence of 5'
gcgtcatgcctaacacatgcaag 3' (SEQ ID NO: 17); (18) an oligonucleotide
having a base sequence of 5' gcgataggcttaacacatgcaag 3' (SEQ ID NO:
18); (19) an oligonucleotide having a base sequence of 5'
gcgacaggcttaacacatgcaag 3' (SEQ ID NO: 19); (20) an oligonucleotide
having a base sequence of 5' gctacaggcttaacacatgcaag 3' (SEQ ID NO:
20); (21) an oligonucleotide having a base sequence of 5'
acagaatgcttaacacatgcaag 3' (SEQ ID NO: 21); (22) an oligonucleotide
having a base sequence of 5' atccagccgcaccttccgatac 3' (SEQ ID NO:
22); (23) an oligonucleotide having a base sequence of 5'
atccaaccgcaggttcccctac 3' (SEQ ID NO: 23); (24) an oligonucleotide
having a base sequence of 5' atccagccgcaggttcccctac 3' (SEQ ID NO:
24); (25) an oligonucleotide having a base sequence of 5'
atccagccgcaccttccggtac 3' (SEQ ID NO: 25); (26) an oligonucleotide
having a base sequence of 5' atccagcgccaggttcccctag 3' (SEQ ID NO:
26); (27) an oligonucleotide having a base sequence of 5'
atccagccgcaggttctcctac 3' (SEQ ID NO: 27); and (28) an
oligonucleotide having a base sequence of 5' atccagccgcacgttcccgtac
3' (SEQ ID NO: 28).
7. A probe set for detecting a gene of infectious disease
pathogenic bacterium, Prevotella denticola, comprising the
following items (A) to (C), wherein the probe set does not comprise
another probe to detect Prevotella denticola: (A) a probe
consisting of the base sequence of TCGATGACGGCATCAGATTCGAAGCA (SEQ
ID NO. 70); (B) a probe consisting of the base sequence of
AATGTAGGCGCCCAACGTCTGACT (SEQ ID NO. 71); and (C) a probe
consisting of the base sequence of ATGTTGAGGTCCTTCGGGACTCCT (SEQ ID
NO. 72).
8. A probe-immobilized carrier according to claim 2, wherein the
first to third probes consist of the following base sequences (A)
to (C) respectively: (A) TCGATGACGGCATCAGATTCGAAGCA (SEQ ID NO.
70); (B) AATGTAGGCGCCCAACGTCTGACT (SEQ ID NO. 71); and (C)
ATGTTGAGGTCCTTCGGGACTCCT (SEQ ID NO. 72).
9. A probe-immobilized carrier according to claim 8, wherein the
probe-immobilized carrier comprises a fourth probe having any one
of the base sequences of SEQ ID NOS. 35 to 94 immobilized at a
position spaced from the first to third probes.
10. A method of detecting a gene of Prevotella denticola in an
analyte by using a probe-immobilized carrier, comprising the steps
of: (i) reacting the analyte with a probe-immobilized carrier
according to claim 2; and (ii) detecting the presence or absence of
a reaction of any one of the first to third probes on the
probe-immobilized carrier with a nucleic acid in the analyte, or
detecting the strength of a hybridization reaction of any one of
the first to third probes on the probe-immobilized carrier with a
nucleic acid in the analyte.
11. A method according to claim 10, further comprising the step of
carrying out PCR amplification of the target nucleic acid in the
analyte by using a primer including the following oligonucleotides:
an oligonucleotide having a base sequence of 5'
gctacaggcttaacacatgcaag 3' (SEQ ID NO: 20); and an oligonucleotide
having a base sequence of 5' atccagccgcaccttccggtac 3' (SEQ ID NO:
25).
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a probe and a probe set for
detecting a gene of infectious disease pathogenic bacterium,
Prevotella denticola, which are useful for detection and
identification of the causative organism of an infectious disease,
a probe-immobilized carrier on which the probe or the probe set is
immobilized, a genetic testing method using the probe-immobilized
carrier, and a genetic testing kit to be used for the method.
2. Description of the Related Art
Heretofore, reagents for and methods of quickly and accurately
detecting the causative organisms of infectious diseases in
analytes have been proposed. For instance, Japanese Patent
Application Laid-Open No. H08-089254 discloses oligonucleotides
having specific base sequences, which can be respectively used as
probes and primers for detecting pathogenic bacteria of candidiasis
and aspergillosis, and a method of detecting target bacteria using
such oligonucleotides. In addition, the same patent document also
discloses a set of primers used for concurrently amplifying a
plurality of target bacteria by PCR. In other words, those primers
are used for the PCR amplification of nucleic acid fragments from
fungi, which serve as a plurality of targets, in an analyte. Target
fungal species in the analyte can be identified by detecting the
presence of a specific part of the sequence by a hybridization
assay using probes specific to the respective fungi and the nucleic
acid fragments amplified by the respective primers.
On the other hand, the method to use probe array in which probes
having sequences complementary to the respective base sequences are
arranged at intervals on a solid support is known as a method
capable of simultaneously detecting a plurality of oligonucleotides
having different base sequences (Japanese Patent Application
Laid-Open No. 2004-313181).
SUMMARY OF THE INVENTION
However, it is no easy task to design a probe for specifically
detecting a gene of an infectious disease pathogenic bacterium in a
sample. That is, as well as the target gene, the sample may further
contain genes of other infectious disease pathogenic bacteria.
Thus, it is no easy task to design the probe that specifically
detects the gene of the infectious disease pathogenic bacterium
while suppressing the cross contamination which is the influence of
the presence of the genes of other infectious disease pathogenic
bacteria. Under such circumstances, the inventors of the present
invention have studied for obtaining a probe which allows accurate
detection of a gene of an infectious disease pathogenic bacterium
as mentioned hereinbelow while maintaining the cross contamination
level low even when a sample in which genes of different bacteria
are present is used. As a result, the inventors of the present
invention have finally found a plurality of probes capable of
precisely detecting the gene of the infectious disease pathogenic
bacterium, Prevotella denticola.
A first object of the present invention is to provide a probe and a
probe set, which can precisely identify a gene of a target
bacterium from an analyte in which various bacteria are
concurrently present. Another object of the present invention is to
provide a probe-immobilized carrier which can be used for precisely
identifying a target bacterium from an analyte in which various
bacteria are concurrently present. Still another object of the
present invention is to provide a genetic testing method for
detecting a target bacterium, which can quickly and precisely
detect the target bacterium from various bacteria in an analyte
when they are present therein, and a kit for such a method.
The probe for detecting a gene of infectious disease pathogenic
bacterium, Prevotella denticola, of the present invention has any
one of the following base sequences (1) to (4):
(1) TCGATGACGGCATCAGATTCGAAGCA (SEQ ID NO. 70) or a complementary
sequence thereof;
(2) AATGTAGGCGCCCAACGTCTGACT (SEQ ID NO. 71) or a complementary
sequence thereof;
(3) ATGTTGAGGTCCTTCGGGACTCCT (SEQ ID NO. 72) or a complementary
sequence thereof; and
(4) a modified sequence prepared such that any one of the sequences
of SEQ ID NOS. 70 to 72 and the complementary sequences thereof is
subjected to base deletion, substitution, or addition as far as the
modified sequence retains a function as the probe.
In addition, the probe set for detecting a gene of infectious
disease pathogenic bacterium, Prevotella denticola, of the present
invention includes at least two probes selected from the following
items (A) to (L):
(A) a probe having a base sequence represented by
TCGATGACGGCATCAGATTCGAAGCA (SEQ ID NO. 70);
(B) a probe having a base sequence represented by
AATGTAGGCGCCCAACGTCTGACT (SEQ ID NO. 71);
(C) a probe having a base sequence represented by
ATGTTGAGGTCCTTCGGGACTCCT (SEQ ID NO. 72);
(D) a probe having a complementary sequence of the base sequence
represented by SEQ ID NO. 70;
(E) a probe having a complementary sequence of the base sequence
represented by SEQ ID NO. 71;
(F) a probe having a complementary sequence of the base sequence
represented by SEQ ID NO. 72;
(G) a probe having a modified sequence obtained by base deletion,
substitution, or addition on the base sequence represented by SEQ
ID NO. 70 as far as it retains the function of a probe for
detecting the gene of Prevotella denticola;
(H) a probe having a modified sequence obtained by base deletion,
substitution, or addition on the base sequence represented by SEQ
ID NO. 71 as far as it retains the function of a probe for
detecting the gene of Prevotella denticola;
(I) a probe having a modified sequence obtained by base deletion,
substitution, or addition on the base sequence represented by SEQ
ID NO. 72 as far as it retains the function of a probe for
detecting the gene of Prevotella denticola;
(J) a probe having a modified sequence obtained by base deletion,
substitution, or addition on the complementary sequence of the base
sequence represented by SEQ ID NO. 70 as far as it retains the
function of a probe for detecting the gene of Prevotella denticola;
(K) a probe having a modified sequence obtained by base deletion,
substitution, or addition on the complementary sequence of the base
sequence represented by SEQ ID NO. 71 as far as it retains the
function of a probe for detecting the gene of Prevotella denticola;
and (L) a probe having a modified sequence obtained by base
deletion, substitution, or addition on the complementary sequence
of the base sequence represented by SEQ ID NO. 72 as far as it
retains the function of a probe for detecting the gene of
Prevotella denticola.
The characteristic feature of the probe-immobilized carrier of the
present invention is that at least one of the above-mentioned
probes (A) to (L) is immobilized on a solid-phase carrier, and when
a plurality of probes are employed, the respective probes are
arranged at intervals.
The method of detecting a gene of an infectious disease pathogenic
bacterium, Prevotella denticola, in an analyte by using a
probe-immobilized carrier of the present invention includes the
steps of:
(i) reacting the analyte with the probe-immobilized carrier having
the above-mentioned constitution; and
(ii) detecting the presence or absence of a reaction of the probe
on the probe-immobilized carrier with a nucleic acid in the
analyte, or detecting the strength of a hybridization reaction of
the probe on the probe-immobilized carrier with a nucleic acid in
the analyte.
The characteristic feature of the kit for detecting an infectious
disease pathogenic bacterium, Prevotella denticola, of the present
invention is to include at least one of the above-mentioned probes
(A) to (L), and a reagent for detecting a reaction between the
probe and a target nucleic acid.
According to the present invention, when an analyte is infected
with the above-mentioned causative bacterium, the bacterium can be
more quickly and precisely identified from the analyte even if the
analyte is simultaneously and complexly infected with other
bacteria in addition to the above-mentioned bacterium. In
particular, Prevotella denticola can be detected while precisely
distinguishing it from Escherichia coli which may otherwise cause
cross contamination.
Further features of the present invention will become apparent from
the following description of exemplary embodiments with reference
to the attached drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagram illustrating a 1st PCR protocol.
FIG. 2 is a diagram illustrating a 2nd PCR protocol.
DESCRIPTION OF THE EMBODIMENTS
The inventors of the present invention have obtained almost all of
bacteria (represented by (1) to (80) below), which have been known
as septicemia pathogenic bacteria so far, from the respective
depository institutions and identified the 16S rRNA gene sequences
of all the bacteria.
Subsequently, while making a comparison of all the identified
sequences, probe sequences for Prevotella denticola were
investigated in detail and the probes of the present invention,
which can identify Prevotella denticola, have finally been found
out.
TABLE-US-00001 (1) Staphylococcus aureus (ATCC12600) (2)
Staphylococcus epidermidis (ATCC14990) (3) Escherichia coil
(ATCC11775) (4) Klebsiella pneumoniae (ATCC13883) (5) Pseudomonas
aeruginosa (ATCC10145) (6) Serratia marcescens (ATCC13380) (7)
Streptococcus pneumoniae (ATCC33400) (8) Haemophilus influenzae
(ATCC33391) (9) Enterobacter cloacae (ATCC13047) (10) Enterococcus
faecalis (ATCC19433) (11) Staphylococcus haemolyticus (ATCC29970)
(12) Staphylococcus hominis (ATCC27844) (13) Staphylococcus
saprophyticus (ATCC15305) (14) Streptococcus agalactiae (ATCC13813)
(15) Streptococcus mutans (ATCC25175) (16) Streptococcus pyogenes
(ATCC12344) (17) Streptococcus sanguinis (ATCC10556) (18)
Enterococcus avium (JCM8722) (19) Enterococcus faecium (ATCC19434)
(20) Pseudomonas fluorescens (ATCC13525) (21) Pseudomonas putida
(ATCC12633) (22) Burkholderia cepacia (JCM5964) (23)
Stenotrophomonas maltophilia (ATCC13637) (24) Acinetobacter
baumannii (ATCC19606) (25) Acinetobacter calcoaceticus (ATCC23055)
(26) Achromobacter xylosoxidans (ATCC27061) (27) Vibrio vulnificus
(ATCC27562) (28) Salmonella choleraesuis (JCM1651) (29) Citrobacter
freundii (ATCC8090) (30) Klebsiella oxytoca (ATCC13182) (31)
Enterobacter aerogenes (ATCC13048) (32) Hafnia alvei (ATCC13337)
(33) Serratia liquefaciens (ATCC27592) (34) Proteus mirabilis
(ATCC29906) (35) Proteus vulgaris (ATCC33420) (36) Morganella
morganii (ATCC25830) (37) Providencia rettgeri (JCM1675) (38)
Aeromonas hydrophila (JCM1027) (39) Aeromonas sobria (ATCC43979)
(40) Gardnerella vaginalis (ATCC14018) (41) Corynebacterium
diphtheriae (ATCC2701) (42) Legionella pneumophila (ATCC33152) (43)
Bacillus cereus (ATCC14579) (44) Bacillus subtilis (ATCC6051) (45)
Mycobacterium kansasii (ATCC12478) (46) Mycobacterium
intracellulare (ATCC13950) (47) Mycobacterium chelonae (ATCC35752)
(48) Nocardia asteroids (ATCC19247) (49) Bacteroides fragilis
(JCM11019) (50) Bacteroides thetaiotaomicron (JCM5827) (51)
Clostridium difficile (ATCC51695) (52) Clostridium perfringens
(JCM1290) (53) Eggerthella lenta (JCM10763) (54) Fusobacterium
necrophorum (JCM3718) (55) Fusobacterium nucleatum (ATCC25586) (56)
Lactobacillus acidophilus (ATCC4356) (57) Anaerococcus prevotii
(JCM6490) (58) Reptoniphilus asaccharolyticus (JCM8143) (59)
Porphyromonas asaccharolytica (JCM6326) (60) Porphyromonas
gingivalis (JCM8525) (61) Prevotella denticola (ATCC38184) (62)
Propionibacterium acnes (JCM6473) (63) Acinetobacter johnsonii
(ATCC17909) (64) Acinetobacter junii (ATCC17908) (65) Aeromonas
schubertii (ATCC43700) (66) Aeromonas veronii (ATCC35624) (67)
Bacteroides distasonis (ATCC8503) (68) Bacteroides vulgatus
(ATCC8482) (69) Campylobacter coli (ATCC33559) (70) Campylobacter
hyointestinalis (ATCC35217) (71) Campylobacter jejuni (ATCC33560)
(72) Flavobacterium aquatile (ATCC11947) (73) Flavobacterium
mizutaii (ATCC33299) (74) Peptococcus niger (ATCC27731) (75)
Propionibacterium avidum (ATCC25577) (76) Propionibacterium
freudenreichii (ATCC6207) (77) Propionibacterium granulosum
(ATCC25564) (78) Clostridium butyricum (ATCC13949) (79)
Flavobacterium hydatis (NBRC14958) (80) Flavobacterium johnsoniae
(NBRC14942)
The deposition numbers of the bacterial species obtained are shown
in the respective parentheses on the right side in the above.
Bacterial species having deposition numbers beginning with "ATCC",
"JCM" and "NBRC" are available from American Type Culture
Collection, Japan Collection of Microorganisms (RIKEN BioResource
Center) and National Board for Respiratory Care, respectively.
The present invention provides an oligonucleotide probe for
identifying an infectious disease pathogenic bacterium
(hereinafter, simply referred to as a probe) and a probe set
including a combination of two or more probes. The use of such a
probe or a probe set allows the detection of the following
bacterium which will cause inflammation by infection.
[Bacterial Name]
Prevotella denticola
That is, the probe of the present invention can detect the 16S rRNA
gene sequence among genes of the above-mentioned bacterium, having
the following sequences:
(A) a probe having a base sequence represented by
TCGATGACGGCATCAGATTCGAAGCA (SEQ ID NO. 70);
(B) a probe having a base sequence represented by
AATGTAGGCGCCCAACGTCTGACT (SEQ ID NO. 71);
(C) a probe having a base sequence represented by
ATGTTGAGGTCCTTCGGGACTCCT (SEQ ID NO. 72);
(D) a probe having a complementary sequence of the base sequence
represented by SEQ ID NO. 70;
(E) a probe having a complementary sequence of the base sequence
represented by SEQ ID NO. 71;
(F) a probe having a complementary sequence of the base sequence
represented by SEQ ID NO. 72;
(G) a probe having a modified sequence obtained by base deletion,
substitution, or addition on the base sequence represented by SEQ
ID NO. 70 as far as it retains the function of a probe for
detecting the gene of Prevotella denticola;
(H) a probe having a modified sequence obtained by base deletion,
substitution, or addition on the base sequence represented by SEQ
ID NO. 71 as far as it retains the function of a probe for
detecting the gene of Prevotella denticola;
(I) a probe having a modified sequence obtained by base deletion,
substitution, or addition on the base sequence represented by SEQ
ID NO. 72 as far as it retains the function of a probe for
detecting the gene of Prevotella denticola;
(J) a probe having a modified sequence obtained by base deletion,
substitution, or addition on the complementary sequence of the base
sequence represented by SEQ ID NO. 70 as far as it retains the
function of a probe for detecting the gene of Prevotella denticola;
(K) a probe having a modified sequence obtained by base deletion,
substitution, or addition on the complementary sequence of the base
sequence represented by SEQ ID NO. 71 as far as it retains the
function of a probe for detecting the gene of Prevotella denticola;
and (L) a probe having a modified sequence obtained by base
deletion, substitution, or addition on the complementary sequence
of the base sequence represented by SEQ ID NO. 72 as far as it
retains the function of a probe for detecting the gene of
Prevotella denticola.
The probe set can be formed using at least two of those probes.
The functions of those probes significantly depend on the
specificity of each probe sequence corresponding to the target
nucleic acid sequence of interest. The specificity of a probe
sequence can be evaluated from the degree of coincidence of bases
with the target nucleic acid sequence and the probe sequence.
Further, when a plurality of probes constitute a probe set, the
variance of melting temperatures among the probes may affect the
performance of the probe set.
For designing a probe sequence, a region showing a high specificity
to a specific bacterial species of interest regardless of any
differences in strain is selected. The region contains three or
more bases which are not coincident with corresponding bases in the
sequences of any other bacterial species. The probe sequence is
designed so that the melting temperature between the probe sequence
and the corresponding sequence of the specific bacterial species of
interest will differ by 10.degree. C. or more from the melting
temperatures between the probe sequence and the corresponding
sequences of any other bacterial species. Further, one or more
bases can be deleted or added so that the respective probes
immobilized on a single carrier may have melting temperatures
within a predetermined range.
The inventors of the present invention found out by experiments
that the hybridization intensity of a probe will not be
significantly attenuated if 80% or more of the base sequence is
consecutively conserved. It can therefore be concluded, from the
finding, such that any sequences modified from the probe sequences
disclosed in the specification will have a sufficient probe
function if 80% or more of the base sequence of the probe is
consecutively conserved.
The above-mentioned modified sequences may include any variation as
far as it does not impair the probe's function, or any variation as
far as it hybridizes with a nucleic acid sequence of interest as a
detection target. Above all, it is desirable to include any
variation as far as it can hybridize with a nucleic acid sequence
of interest as a detection target under stringent conditions.
Preferable hybridization conditions confining the variation include
those represented in examples as described below. Here, the term
"detection target" used herein may be one included in a sample to
be used in hybridization, which may be a unique base sequence to
the infectious disease pathogenic bacterium, or may be a
complementary sequence to the unique sequence. Further, the
variation may be a modified sequence obtained by deletion,
substitution, or addition of at least one base as far as it retains
a function as the probe.
Those probe sequences are only specific to the DNA sequence coding
for the 16S rRNA of the above-mentioned bacterium, so sufficient
hybridization sensitivity to the sequence will be expected even
under stringent conditions. In addition, any of those probe
sequences forms a stable hybridized product through a hybridization
reaction thereof with a target analyte even when the probe
sequences are immobilized on a carrier, which is designed to
produce an excellent result.
Further, a probe-immobilized carrier (e.g., DNA chip), on which the
probe for detecting the infectious disease pathogenic bacterium of
the present invention, can be obtained by supplying the probe on a
predetermined position on the carrier and immobilizing the probe
thereon. Various methods can be used for supplying the probe to the
carrier. Among them, for example, a method, which can be suitably
used, is to keep a surface state capable of immobilizing the probe
on the carrier through a chemical bonding (e.g., covalent bonding)
and a liquid containing the probe is then provided on a
predetermined position by an inkjet method. Such a method allows
the probe to be hardly detached from the carrier and exerts an
additional effect of improving the sensitivity. In other words,
when a stamping method conventionally used and called the Stanford
method is employed to make a DNA chip, the resultant DNA chip has a
disadvantage such that the applied DNA tends to be peeled off.
Another one of the methods of forming DNA chips is to carry out the
arrangement of probes by the synthesis of DNA on the surface of a
carrier (e.g., DNA chip from Affymetrix Co., Ltd.). In such a
method of synthesizing probes on a carrier, it is difficult to make
equal the amount of synthesized DNA for each probe sequence. Thus,
the amount of immobilized probe per immobilization area (spot) for
each probe tends to vary considerably. Such variations in amounts
of the respective immobilized probes may cause incorrect evaluation
on the results of the detection with those probes. Based on this
fact, the probe carrier of the present invention is preferably
prepared using the above-mentioned inkjet method. The inkjet method
as described above has an advantage such that the probe can be
stably immobilized on the carrier and hardly detaching from the
carrier to efficiently provide a probe carrier which can carry out
detection with high sensitivity and high accuracy.
In addition, a probe set may include at least two selected from the
group consisting of SEQ ID NOS. 70 to 72 as described above and the
complementary sequences thereof and sequences obtained by base
deletion, substitution, or addition on those sequences as far as
they retain the function of a probe for detecting the gene of
Prevotella denticola. In this case, the accuracy of detecting the
Prevotella denticola gene can be further improved.
Hereinafter, preferred embodiments of the present invention will be
described in detail.
Test objects to be tested using probe carriers (e.g., DNA chips) in
which the probes of the present invention are immobilized on
carriers include those originated from humans and animals such as
domestic animals. For example, a test object is any of those which
may contain bacteria, including: any body fluids such as blood,
cerebrospinal fluid, expectorated sputum, gastric juice, vaginal
discharge, and oral mucosal fluid; and excretions such as urine and
feces. All media, which can be contaminated with bacteria, can be
also subjected to a test using a DNA chip. Such media include:
food, drink water and water in the natural environment such as hot
spring water, which may cause food poisoning by contamination;
filters of air cleaners and the like; and so on. Animals and
plants, which should be quarantined in import/export, are also used
as analytes of interest.
When the sample as described above can be directly used in reaction
with the DNA chip, it is used as an analyte to react with the DNA
chip and the result of the reaction is then analyzed.
Alternatively, when the sample cannot be directly reacted with the
DNA chip, the sample was subjected to extraction, purification, and
other procedures for obtaining a target substance if required and
then provided as an analyte to carry out a reaction with the DNA
chip. For instance, when the sample contains a target nucleic acid,
an extract, which may be assumed to contain such a target nucleic
acid, is prepared from a sample, and then washed, diluted, or the
like to obtain an analyte solution followed by reaction with the
DNA chip. Further, as a target nucleic acid is included in an
analyte obtained by carrying out various amplification procedures
such as PCR amplification, the target nucleic acid may be amplified
and then reacted with a DNA chip. Such analytes of amplified
nucleic acids include the following ones:
(a) An amplified analyte prepared by using a PCR-reaction primer
designed for detecting 16S rRNA gene.
(b) An amplified analyte prepared by an additional PCR reaction or
the like from a PCR-amplified product.
(c) An analyte prepared by an amplification method other than
PCR.
(d) An analyte labeled for visualization by any of various labeling
methods.
Further, a carrier used for preparing a probe-immobilized carrier,
such as a DNA chip, may be any of those that satisfy the property
of carrying out a solid phase/liquid phase reaction of interest.
Examples of the carrier include: flat substrates such as a glass
substrate, a plastic substrate, and a silicon wafer; a
three-dimensional structure having an irregular surface; and a
spherical body such as a bead, and rod-, cord-, and thread-shaped
structures. The surface of the carrier may be processed such that a
probe can be immobilized thereon. Especially, a carrier prepared by
introducing a functional group to its surface to enable chemical
reaction has a preferable form from the viewpoint of
reproducibility because the probe is stably bonded in the process
of hybridization reaction.
Various methods can be employed for the immobilization of probes.
An example of such a method is to use a combination of a maleimide
group and a thiol (--SH) group. In this method, a thiol (--SH)
group is bonded to the terminal of a probe, and a process is
executed in advance to make the carrier (solid) surface have a
maleimide group. Accordingly, the thiol group of the probe supplied
to the carrier surface reacts with the maleimide group on the
carrier surface to form a covalent bond, whereby the probe is
immobilized.
Introduction of the maleimide group can utilize a process of
firstly allowing a reaction between a glass substrate and an
aminosilane coupling agent and then introducing a maleimide group
onto the glass substrate by a reaction of the amino group with an
EMCS reagent (N-(6-maleimidocaproyloxy)succinimide, available from
Dojindo). Introduction of the thiol group to a DNA can be carried
out using 5'-Thiol-Modifier C6 (available from Glen Research) when
the DNA is synthesized by an automatic DNA synthesizer.
Instead of the above-described combination of a thiol group and a
maleimide group, a combination of, e.g., an epoxy group (on the
solid phase) and an amino group (nucleic acid probe terminal), can
also be used as a combination of functional groups to be used for
immobilization. Surface treatments using various kinds of silane
coupling agents are also effective. A probe in which a functional
group which can react with a functional group introduced by a
silane coupling agent is introduced is used. A method of applying a
resin having a functional group can also be used.
The detection of the gene of the infectious disease pathogenic
bacterium by using the probe-immobilized carrier of the present
invention can be carried out by a genetic testing method including
the steps of:
(i) reacting an analyte with a probe-immobilized carrier on which
the probe of the present invention is immobilized;
(ii) detecting the presence or absence of the reaction of a nucleic
acid in the analyte with the probe on the probe-immobilized
carrier, or detecting the strength of the hybridization reaction of
a nucleic acid in the analyte with the probe on the
probe-immobilized carrier; and (iii) specifying the probe having
reacted with the nucleic acid in the analyte when the reaction of
the probe with the nucleic acid in the analyte is detected and
specifying the gene of the infectious disease pathogenic bacterium
in the analyte based on the nucleic acid sequence of the probe.
The probe to be immobilized on the probe-immobilized carrier is at
least one of the above-mentioned items (A) to (L). On the carrier,
probes for detecting bacterial species other than Prevotella
denticola may be immobilized as other probes, depending on the
purpose of test. In this case, the other probes may be those
capable of detecting the bacterial species other than Prevotella
denticola without causing cross contamination and the use of such
probes allows simultaneous detection of a plurality of bacterial
species with high accuracy.
Further, as described above, when the 16S rRNA gene sequence of an
infectious disease pathogenic bacterium in the analyte is amplified
by PCR and provided as a sample to be reacted with a probe carrier,
a primer set for detecting the infectious disease pathogenic
bacterium can be used. The primer set suitably includes at least
one selected from oligonucleotides represented in the following
items (1) to (21) and at least one selected from oligonucleotides
represented in the following items (22) to (28), more suitably
includes all the oligonucleotides represented in the following
items (1) to (28):
(1) an oligonucleotide having a base sequence of 5'
gcggcgtgcctaatacatgcaag 3' (SEQ ID NO: 1);
(2) an oligonucleotide having a base sequence of 5'
gcggcaggcctaacacatgcaag 3' (SEQ ID NO: 2);
(3) an oligonucleotide having a base sequence of 5'
gcggcaggcttaacacatgcaag 3' (SEQ ID NO: 3);
(4) an oligonucleotide having a base sequence of 5'
gcggtaggcctaacacatgcaag 3' (SEQ ID NO: 4);
(5) an oligonucleotide having a base sequence of 5'
gcggcgtgcttaacacatgcaag 3' (SEQ ID NO: 5);
(6) an oligonucleotide having a base sequence of 5'
gcgggatgccttacacatgcaag 3' (SEQ ID NO: 6);
(7) an oligonucleotide having a base sequence of 5'
gcggcatgccttacacatgcaag 3' (SEQ ID NO: 7);
(8) an oligonucleotide having a base sequence of 5'
gcggcatgcttaacacatgcaag 3' (SEQ ID NO: 8);
(9) an oligonucleotide having a base sequence of 5'
gcggcgtgcttaatacatgcaag 3' (SEQ ID NO: 9);
(10) an oligonucleotide having a base sequence of 5'
gcggcaggcctaatacatgcaag 3' (SEQ ID NO: 10);
(11) an oligonucleotide having a base sequence of 5'
gcgggatgctttacacatgcaag 3' (SEQ ID NO: 11);
(12) an oligonucleotide having a base sequence of 5'
gcggcgtgcctaacacatgcaag 3' (SEQ ID NO: 12);
(13) an oligonucleotide having a base sequence of 5'
gcggcgtgcataacacatgcaag 3' (SEQ ID NO: 13);
(14) an oligonucleotide having a base sequence of 5'
gcggcatgcctaacacatgcaag 3' (SEQ ID NO: 14);
(15) an oligonucleotide having a base sequence of 5'
gcggcgcgcctaacacatgcaag 3' (SEQ ID NO: 15);
(16) an oligonucleotide having a base sequence of 5'
gcggcgcgcttaacacatgcaag 3' (SEQ ID NO: 16);
(17) an oligonucleotide having a base sequence of 5'
gcgtcatgcctaacacatgcaag 3' (SEQ ID NO: 17);
(18) an oligonucleotide having a base sequence of 5'
gcgataggcttaacacatgcaag 3' (SEQ ID NO: 18);
(19) an oligonucleotide having a base sequence of 5'
gcgacaggcttaacacatgcaag 3' (SEQ ID NO: 19);
(20) an oligonucleotide having a base sequence of 5'
gctacaggcttaacacatgcaag 3' (SEQ ID NO: 20);
(21) an oligonucleotide having a base sequence of 5'
acagaatgcttaacacatgcaag 3' (SEQ ID NO: 21);
(22) an oligonucleotide having a base sequence of 5'
atccagccgcaccttccgatac 3' (SEQ ID NO: 22);
(23) an oligonucleotide having a base sequence of 5'
atccaaccgcaggttcccctac 3' (SEQ ID NO: 23);
(24) an oligonucleotide having a base sequence of 5'
atccagccgcaggttcccctac 3' (SEQ ID NO: 24);
(25) an oligonucleotide having a base sequence of 5'
atccagccgcaccttccggtac 3' (SEQ ID NO: 25);
(26) an oligonucleotide having a base sequence of 5'
atccagcgccaggttcccctag 3' (SEQ ID NO: 26);
(27) an oligonucleotide having a base sequence of 5'
atccagccgcaggttctcctac 3' (SEQ ID NO: 27); and
(28) an oligonucleotide having a base sequence of 5'
atccagccgcacgttcccgtac 3' (SEQ ID NO: 28).
Among them, a primer designed for allowing the amplification of
Prevotella denticola is a primer set of the following:
(20) an oligonucleotide having a base sequence of 5'
gctacaggcttaacacatgcaag 3' (SEQ ID NO: 20); and
(25) an oligonucleotide having a base sequence of 5'
atccagccgcaccttccggtac 3' (SEQ ID NO: 25).
For detecting Prevotella denticola, at least such a primer may be
included.
The utilities of the respective primers (1) to (28) for
amplification of Prevotella denticola (ATCC 38184) can be evaluated
and confirmed by comparing each sequence of SEQ ID NOs. 1 to 28
with a DNA sequence including the 16S rRNA coding region of
Prevotella denticola (SEQ ID NO. 95).
A kit for detecting the infectious disease pathogenic bacterium can
be constructed using at least a probe as described above and a
reagent for detecting a reaction of the probe with a nucleic acid
in an analyte. The probe in the kit can preferably be provided as a
probe-immobilized carrier as described above. Further, the
detection reagent may contain a label to detect the reaction or a
primer for carrying out amplification as a pre-treatment.
EXAMPLES
Hereinafter, the present invention will be described in more detail
with reference to examples using probes for detecting an infectious
disease pathogenic bacterium to detect Prevotella denticola.
Example 1
In this example, microorganism detection using 2-step PCR will be
described.
1. Preparation of Probe DNA
Nucleic acid sequences shown in Table 1 were designed as probes to
be used for detection of Prevotella denticola. Specifically, the
following probe base sequences were selected from the genome part
coding for the 16s rRNA gene of Prevotella denticola. These probe
base sequences were designed such that they could have an extremely
high specificity to the bacterium, and a sufficient hybridization
sensitivity could be expected without variance for the respective
probe base sequences. The probe base sequences need not always
completely match with those shown in Table 1. Probes having base
lengths of 20 to 30 which include the base sequences shown in Table
1 can also be used, in addition to the probes having the base
sequences shown in Table 1. However, it should be ensured that the
other portion of the base sequence than the portion shown in Table
1 in such a probe has no effect on the detection accuracy.
TABLE-US-00002 TABLE 1 Name of microorganism Prevotella denticola
SEQ ID Probe No. NO. Sequence 061_P_den_01 70 5'
TCGATGACGGCATCAGATTCGAAGCA 3' 061_P_den_02 71 5'
AATGTAGGCGCCCAACGTCTGACT 3' 061_P_den_03 72 5'
ATGTTGAGGTCCTTCGGGACTCCT 3'
For each probe having a base sequence shown in Table 1, a thiol
group was introduced, as a functional group to immobilize the probe
on a DNA chip, to the 5' terminal of the nucleic acid after
synthesis in accordance with a conventional method. After
introduction of the functional group, purification and
freeze-drying were executed. The freeze-dried probes for internal
standard were stored in a freezer at -30.degree. C.
2. Preparation of PCR Primers
2-1. Preparation of PCR Primers for Analyte Amplification
As 16S rRNA gene (target gene) amplification PCR primers for
pathogenic bacterium detection, nucleic acid sequences shown in
Table 2 below were designed. Specifically, primer sets which
specifically amplify the genome parts coding the 16S rRNAs, i.e.,
primers for which the specific melting points were made uniform as
far as possible at the two end portions of the 16S rRNA coding
region of a base length of 1,400 to 1,700 were designed. In order
to simultaneously amplify a plurality of different bacterial
species listed in the following items (1) to (80), mutants, or a
plurality of 16S rRNA genes on genomes, a plurality of kinds of
primers were designed. Note that a primer set is not limited to the
primer sets shown in Table 2 as far as the primer set is available
in common to amplify almost the entire lengths of the 16S rRNA
genes of the pathogenic bacteria.
TABLE-US-00003 TABLE 2 Primer No SEQ ID NO. Sequence F01 1 5'
GCGGCGTGCCTAATACATGCAAG 3' F02 2 5' GCGGCAGGCCTAACACATGCAAG 3' F03
3 5' GCGGCAGGCTTAACACATGCAAG 3' F04 4 5' GCGGTAGGCCTAACACATGCAAG 3'
F05 5 5' GCGGCGTGCTTAACACATGCAAG 3' F06 6 5'
GCGGGATGCCTTACACATGCAAG 3' F07 7 5' GCGGCATGCCTTACACATGCAAG 3' F08
8 5' GCCGCATGCTTAACACATGCAAG 3' F09 9 5' GCGGCGTGCTTAATACATGCAAG 3'
F10 10 5' GCGGCAGGCCTAATACATGCAAG 3' F11 11 5'
GCGGGATGCTTTACACATGCAAG 3' F12 12 5' GCGGCGTGCCTAACACATGCAAG 3' F13
13 5' GCGGCGTGCATAACACATGCAAG 3' F14 14 5' GCGGCATGCCTAACACATGCAAG
3' F15 15 5' GCGGCGCGCCTAACACATGCAAG 3' F16 16 5'
GCGCCGCGCTTAACACATGCAAG 3' F17 17 5' GCGTCATGCCTAACACATGCAAG 3' F18
18 5' GCGATAGGCTTAACACATGCAAG 3' F19 19 5' GCGACAGGCTTAACACATGCAAG
3' F20 20 5' GCTACAGGCTTAACACATGCAAG 3' F21 21 5'
ACAGAATGCTTAACACATGCAAG 3' R01 22 5' ATCCAGCCGCACCTTCCGATAC 3' R02
23 5' ATCCAACCGCAGGTTCCCCTAC 3' R03 24 5' ATCCAGCCGCAGGTTCCCCTAC 3'
R04 25 5' ATCCAGCCGCACCTTCCGGTAC 3' R05 26 5'
ATCCAGCGCCAGGTTCCCCTAG 3' R06 27 5' ATCCAGCCGCAGGTTCTCCTAC 3' R07
28 5' ATCCAGCCGCACGTTCCCGTAC 3'
(1) Staphylococcus aureus
(2) Staphylococcus epidermidis
(3) Escherichia coli
(4) Klebsiella pneumoniae
(5) Pseudomonas aeruginosa
(6) Serratia marcescens
(7) Streptococcus pneumoniae
(8) Haemophilus influenzae
(9) Enterobacter cloacae
(10) Enterococcus faecalis
(11) Staphylococcus haemolyticus
(12) Staphylococcus hominis
(13) Staphylococcus saprophyticus
(14) Streptococcus agalactiae
(15) Streptococcus mutans
(16) Streptococcus pyogenes
(17) Streptococcus sanguinis
(18) Enterococcus avium
(19) Enterococcus faecium
(20) Pseudomonas fluorescens
(21) Pseudomonas putida
(22) Burkholderia cepacia
(23) Stenotrophomonas maltophilia
(24) Acinetobacter baumannii
(25) Acinetobacter calcoaceticus
(26) Achromobacter xylosoxidans
(27) Vibrio vulnificus
(28) Salmonella choleraesuis
(29) Citrobacter freundii
(30) Klebsiella oxytoca
(31) Enterobacter aerogenes
(32) Hafnia alvei
(33) Serratia liquefaciens
(34) Proteus mirabilis
(35) Proteus vulgaris
(36) Morganella morganii
(37) Providencia rettgeri
(38) Aeromonas hydrophila
(39) Aeromonas sobria
(40) Gardnerella vaginalis
(41) Corynebacterium diphtheriae
(42) Legionella pneumophila
(43) Bacillus cereus
(44) Bacillus subtilis
(45) Mycobacterium kansasii
(46) Mycobacterium intracellulare
(47) Mycobacterium chelonae
(48) Nocardia asteroides
(49) Bacteroides fragilis
(50) Bacteroides thetaiotaomicron
(51) Clostridium difficile
(52) Clostridium perfringens
(53) Eggerthella lenta
(54) Fusobacterium necrophorum
(55) Fusobacterium nucleatum
(56) Lactobacillus acidophilus
(57) Anaerococcus prevotii
(58) Peptoniphilus asaccharolyticus
(59) Porphyromonas asaccharolytica
(60) Porphyromonas gingivalis
(61) Prevotella denticola
(62) Propionibacterium acnes
(63) Acinetobacter johnsonii
(64) Acinetobacter junii
(65) Aeromonas schubertii
(66) Aeromonas veronii
(67) Bacteroides distasonis
(68) Bacteroides vulgatus
(69) Campylobacter coli
(70) Campylobacter hyointestinalis
(71) Campylobacter jejuni
(72) Flavobacterium aquatile
(73) Flavobacterium mizutaii
(74) Peptococcus niger
(75) Propionibacterium avidum
(76) Propionibacterium freudenreichii
(77) Propionibacterium granulosum
(78) Clostridium butyricum
(79) Flavobacterium hydatis
(80) Flavobacterium johnsoniae
The primers shown in Table 2 were purified by high performance
liquid chromatography (HPLC) after synthesis. The twenty-one
forward primers and the seven reverse primers were mixed and
dissolved in a TE buffer solution such that each primer
concentration had an ultimate concentration of 10 pmol/.mu.l.
2-2. Preparation of Labeling PCR Primers
In a manner similar to the above-mentioned analyte amplification
primers, oligonucleotides having sequences as shown in Table 3
below were employed as primers for labeling.
TABLE-US-00004 TABLE 3 Primer No SEQ ID NO. Sequence Cy3R9-1 29 5'
TACCTTGTTACGACTTCACCCCA 3' Cy3R9-2 30 5' TACCTTGTTACGACTTCGTCCCA 3'
Cy3R9-3 31 5' TACCTTGTTACGACTTAGTCCCA 3' Cy3R9-4 32 5'
TACCTTGTTACGACTTAGCCCCA 3' Cy3R9-5 33 5' TACCTTGTTACGACTTAGTCCTA 3'
Cy3R9-6 34 5' TACCTTGTTACGACTTAGCCCTA 3'
The primers shown in Table 3 were labeled with a fluorescent dye,
Cy3. The primers were purified by high performance liquid
chromatography (HPLC) after synthesis. The six labeled primers were
mixed and dissolved in a TE buffer solution such that each primer
concentration had an ultimate concentration of 10 pmol/.mu.l.
3. Extraction of Genome DNA (Model Analyte) of Prevotella
denticola
3-1. Microbial Culture & Genome DNA Extraction
First, Prevotella denticola (ATCC 38184) was cultured in accordance
with the conventional method. This microbial culture medium was
subjected to the extraction and purification of genome DNA by using
a nucleic acid purification kit (FastPrep FP100A FastDNA Kit,
manufactured by Funakoshi Co., Ltd.).
3-2. Test of Collected Genome DNA
The collected genome DNA of the microorganism, Prevotella
denticola, was subjected to agarose electrophoresis and 260/280-nm
absorbance determination in accordance with the conventional
method. Thus, the quality (the admixture amount of low molecular
nucleic acid and the degree of decomposition) and the collection
amount were tested. In this example, about 10 .mu.g of the genome
DNA was collected. No degradation of genome DNA or contamination of
rRNA was observed. The collected genome DNA was dissolved in a TE
buffer solution at an ultimate concentration of 50 ng/.mu.l and
used in the following experiments.
4. Preparation of DNA Chip
4-1. Cleaning of Glass Substrate
A glass substrate (size: 25 mm.times.75 mm.times.1 mm, available
from Iiyama Precision Glass) made of synthetic quartz was placed in
a heat- and alkali-resisting rack and dipped in a cleaning solution
for ultrasonic cleaning, which was adjusted to have a predetermined
concentration. The glass substrate was kept dipped in the cleaning
solution for a night and cleaned by ultrasonic cleaning for 20 min.
The substrate was picked up, lightly rinsed with pure water, and
cleaned by ultrasonic cleaning in ultrapure water for 20 min. The
substrate was dipped in a 1N aqueous sodium hydroxide solution
heated to 80.degree. C. for 10 min. Pure water cleaning and
ultrapure water cleaning were executed again. A quartz glass
substrate for a DNA chip was thus prepared.
4-2. Surface Treatment
A silane coupling agent KBM-603 (available from Shin-Etsu Silicone)
was dissolved in pure water at a concentration of 1% by weight (wt
%) and stirred at room temperature for 2 hrs. The cleaned glass
substrate was dipped in the aqueous solution of the silane coupling
agent and left stand still at room temperature for 20 min. The
glass substrate was picked up. The surface thereof was lightly
rinsed with pure water and dried by spraying nitrogen gas to both
surfaces of the substrate. The dried substrate was baked in an oven
at 120.degree. C. for 1 hr to complete the coupling agent
treatment, whereby an amino group was introduced to the substrate
surface. Next, N-maleimidocaproyloxy succinimido (abbreviated as
EMCS hereinafter) was dissolved in a 1:1 (volume ratio) solvent
mixture of dimethyl sulfoxide and ethanol to obtain an ultimate
concentration of 0.3 mg/ml. As a result, an EMCS solution was
prepared. Here, EMCS is N-(6-maleimidocaproyloxy)succinimido
available from Dojindo.
The baked glass substrate was left stand and cooled and dipped in
the prepared EMCS solution at room temperature for 2 hrs. By this
treatment, the amino group introduced to the surface of the
substrate by the silane coupling agent reacted with the succinimide
group in the EMCS to introduce the maleimide group to the surface
of the glass substrate. The glass substrate picked up from the EMCS
solution was cleaned by using the above-described solvent mixture
in which the EMCS was dissolved. The glass substrate was further
cleaned by ethanol and dried in a nitrogen gas atmosphere.
4-3. Probe DNA
The microorganism detection probe prepared in the stage 1
(Preparation of Probe DNA) of Example 1 was dissolved in pure
water. The solution was dispensed such that the ultimate
concentration (at ink dissolution) became 10 .mu.M. Then, the
solution was freeze-dried to remove water
4-4. DNA Discharge by BJ Printer and Bonding to Substrate
An aqueous solution containing 7.5-wt % glycerin, 7.5-wt %
thiodiglycol, 7.5-wt % urea, and 1.0-wt % Acetylenol EH (available
from Kawaken Fine Chemicals) was prepared. Each of the three probes
(Table 1) prepared in advance was dissolved in the solvent mixture
at a specific concentration. An ink tank for an inkjet printer
(trade name: BJF-850, available from Canon) is filled with the
resultant DNA solution and attached to the printhead.
The inkjet printer used here was modified in advance to allow
printing on a flat plate. When a printing pattern is input in
accordance with a predetermined file creation method, a about
5-picoliter of a DNA solution can be spotted at a pitch of about
120 .mu.m.
The printing operation was executed for one glass substrate by
using the modified inkjet printer to prepare an array. After
confirming that printing was reliably executed, the glass substrate
was left stand still in a humidified chamber for 30 min to make the
maleimide group on the glass substrate surface react with the thiol
group at the nucleic acid probe terminal
4-5. Cleaning
After reaction for 30 min, the DNA solution remaining on the
surface was cleaned by using a 10-mM phosphate buffer (pH 7.0)
containing 100-mM NaCl, thereby obtaining a DNA chip in which
single-stranded DNAs were immobilized on the glass substrate
surface
5. Amplification and Labeling of Analyt
5-1. Amplification of Analyte: 1st PCR
The amplification reaction (1st PCR) and the labeling reaction (2nd
PCR) of a microbial gene to be provided as an analyte are shown in
Table 4 below.
TABLE-US-00005 TABLE 4 AmpliTaq Gold LD (5.0 U/.mu.L) 0.5 .mu.L
(2.5 unit/tube) Primer mix <FR21x7> 2.0 .mu.L Forward primer
(x21 [0.625 .mu.M/each]) (final 1.25 pmol each/tube) Reverse primer
(x07 [1.875 .mu.M/each]) (final 3.75 pmol each/tube) 10x PCR buffer
5.0 .mu.L (final 1x conc.) MgCl.sub.2 (25 mM) 8.0 .mu.L (final 4.0
mM) dNTPmix (2.5 mM/each) 4.0 .mu.L (final 200 .mu.M each) Template
variable H.sub.2O up to 50 .mu.L Total 50 .mu.L
Amplification reaction of the reaction solution having the
above-mentioned composition was carried out using a commercially
available thermal cycler in accordance with the protocol
illustrated in FIG. 1. After the end of reaction, the primer was
purified using a purification column (QIAquick PCR Purification Kit
available from QIAGEN). Subsequently, the quantitative assay of the
amplified product was carried out.
5-2. Labeling Reaction: 2nd PCR
Amplification reaction of the reaction solution having the
composition shown in Table 5 was carried out using a commercially
available thermal cycler in accordance with the protocol
illustrated in FIG. 2.
TABLE-US-00006 TABLE 5 Premix Taq (Ex Taq Version) 25 .mu.L
Cy3-labeled reverse primer mix 0.83 .mu.L Cy3R9 mix (x06[10
.mu.M/each]) (final 8.3 pmol each/tube) Template variable (final 30
ng/tube) H.sub.2O up to 50 .mu.L Total 50 .mu.L
After the end of reaction, the primer was purified using a
purification column (QIAquick PCR Purification Kit available from
QIAGEN) to obtain a labeled analyte.
6. Hybridization
Detection reaction was performed using the DNA chip prepared in the
stage 4 (Preparation of DNA Chip) and the labeled analyte prepared
in the stage 5 (Amplification and Labeling of Analyte).
6-1. Blocking of DNA Chip
Bovine serum albumin (BSA, Fraction V: available from Sigma) was
dissolved in a 100-mM NaCl/10-mM phosphate buffer such that a 1 wt
% solution was obtained. Then, the DNA chip prepared in the stage 4
(Preparation of DNA Chip) was dipped in the solution at room
temperature for 2 hrs to execute blocking. After the end of
blocking, the chip was cleaned using a washing solution as
described below, rinsed with pure water and hydro-extracted by a
spin dryer.
The washing solution: 2.times.SSC solution (NaCl-300 mM, sodium
citrate (trisodium citrate dihydrate,
C.sub.6H.sub.5Na.sub.3.2H.sub.2O) 30 mM, pH 7.0) containing 0.1-wt
% sodium dodecyl sulfate (SDS)
6-2. Hybridization
The hydro-extracted DNA chip was placed in a hybridization
apparatus (Hybridization Station available from Genomic Solutions
Inc). Hybridization reaction was carried out in a hybridization
solution under conditions as described below.
6-3. Hybridization Solution
6.times.SSPE/10% formamide/target (all 2nd PCR products)/0.05 wt %
(6.times.SSPE: NaCl 900 mM, NaH.sub.2PO.sub.4H.sub.2O 50 mM, EDTA 6
mM, pH, 7.4)
6-4. Hybridization Conditions
65.degree. C. for 3 min, 55.degree. C. for 4 hrs, washing with
2.times.SSC/0.1% SDS at 50.degree. C., washing with
2.times.SSC/0.1% SDS at 20.degree. C. (rinse with H.sub.2O:
manual), and spin dry.
7. Microorganism Genome Detection (Fluorometry)
The DNA chip after the end of hybridization reaction was subjected
to fluorometry with a DNA chip fluorescent detector (GenePix 4000B
available from Axon). As a result, Prevotella denticola was able to
be detected with a sufficient signal at a high reproducibility. The
results of fluorometry are shown in Table 6 below.
TABLE-US-00007 TABLE 6 Fluorescence intensity Probe No. Prevotella
denticola (ATCC 38184) 061_P_den_01 3770.2 061_P_den_02 5472.5
061_P_den_03 3902.4
8. Hybridization with Other Bacterial Species
For proving the fact that the probe set shown in Table 1 can be
specifically hybridized only with Prevotella denticola, the results
of hybridization reaction with Escherichia coli (JCM 1649) are
shown in Table 7 below.
TABLE-US-00008 TABLE 7 Fluorescence intensity Probe No. Escherichia
coli (JCM 1649) 061_P_den_01 50.1 061_P_den_02 50.1 061_P_den_03
50.1
9. Results
As is evident from the above description, a DNA chip was prepared
such that a probe set, which was able to detect only Prevotella
denticola in a specific manner, was immobilized. Further, the use
of such a DNA chip allowed the identification of an infectious
disease pathogenic bacterium, so the problems of the DNA probe
derived from a microorganism was able to be solved. In other words,
the oligonucleotide probe can be chemically produced in large
amounts, while the purification or concentration thereof can be
controlled. In addition, for classification of microbial species, a
probe set capable of collectively detecting bacterial strains of
the same genus and differentially detecting them from bacteria of
other genera, was able to be provided.
Further, in addition to Escherichia coli as described above,
hybridization reaction was carried out on each of nucleic acids
extracted from the bacteria represented in the above-mentioned
items (1) to (80). The results thereof confirmed that no
substantial reaction was observed with respect to each of those
bacteria in a manner similar to that of Escherichia coli, except of
Prevotella denticola.
The bacteria represented in the above-mentioned items (1) to (80)
are pathogenic bacteria for septicemia, and they cover almost all
of the pathogenic bacteria ever detected in human blood. Therefore,
by using the primer of the present embodiment, the nucleic acid of
an infectious disease pathogenic bacterium in blood can be
extracted and then subjected to hybridization reaction with the
probe of the present invention, whereby identification of
Prevotella denticola can be performed with higher accuracy.
Further, according to the above-mentioned example, the presence of
an infectious disease pathogenic bacterium can be efficiently
determined with high accuracy by completely detecting the 16S rRNA
gene from the gene of the infectious disease pathogenic
bacterium.
Example 2
Preparation of DNA Chip by which Various Bacterial Species can be
Simultaneously Determined
In a manner similar to the stage 1 (Preparation of Probe DNA) of
Example 1, probes having base sequences as shown in Table 8 below
were prepared.
TABLE-US-00009 TABLE 8 Bacterial species (or SEQ ID genus) of
interest Probe sequence (5' .fwdarw. 3') NO. Anaerococcus prevotii
TCATCTTGAGGTATGGAAGGGAAAGTGG 35 GTGTTAGGTGTCTGGAATAATCTGGGTG 36
ACCAAGTCTTGACATATTACGGCGG 37 Bacteroides fragilis
AAGGATTCCGGTAAAGGATGGGGATG 38 TGGAAACATGTCAGTGAGCAATCACC 39
Bacteroides AAGAATTTCGGTTATCGATGGGGATGC 40 thetaiotaomicron
AAGTTTTCCACGTGTGGAATTTTGTATGT 41 AAGGCAGCTACCTGGTGACAGGAT 42
Clostridium difficile AATATCAAAGGTGAGCCAGTACAGGATGGA 43
CCGTAGTAAGCTCTTGAAACTGGGAGAC 44 TCCCAATGACATCTCCTTAATCGGAGAG 45
Clostridium perfringens AACCAAAGGAGCAATCCGCTATGAGAT 46
GAGCGTAGGCGGATGATTAAGTGGG 47 CCCTTGCATTACTCTTAATCGAGGAAATC 48
Eggerthella lenta GGAAAGCCCAGACGGCAAGGGA 49
CCTCTCAAGCGGGATCTCTAATCCGA 50 TGCCCCATGTTGCCAGCATTAGG 51
Fusobacterium necrophorum TTTTCGCATGGAGGAATCATGAAAGCTA 52
GATGCGCCGGTGCCCTTTCG 53 GTCGGGAAGAAGTCAGTGACGGTAC 54 Peptoniphilus
GAGTACGTGCGCAAGCATGAAACT 55 asaccharolyticus Porphyromonas
GAAGACTGCCCGCAAGGGTTGTAA 56 asaccharolytica
GTGTACTGGAGGTACGTGGAACGTG 57 GCATGAGGCTGAGAGGTCTCTTCC 58
Porphyromonas gingivalis TTATAGCTGTAAGATAGGCATGCGTCCC 59
AACGGGCGATACGAGTATTGCATTGA 60 ATATACCGTCAAGCTTCCACAGCGA 61
Enterococcus avium TTTGAAAGGCGCTTTTGCGTCACTG 62
CAAGGATGAGAGTAGAACGTTCATCCCTTG 63 CAAGGATGAGAGTAGAATGTTCATCCCTTG 64
Providencia rettgeri CCTGGGAATGGCATCTAAGACTGGTCA 65 Acinetobacter
(genus) GAGGAAGGCGTTGATGCTAATATCATCA 66 GAGCAAAGCAGGGGAACTTCGGTC 67
GTTGGGGCCTTTGAGGCTTTAGTG 68 TGGGAGAGGATGGTAGAATTCCAGGT 69
Prevotella denticola TCGATGACGGCATCAGATTCGAAGCA 70
AATGTAGGCGCCCAACGTCTGACT 71 ATGTTGAGGTCCTTCGGGACTCCT 72
Flavobacterium (genus) GGAAGTAACTAGAATATGTAGTGTAGCGGTG 73
GCCAGTGCAAACTGTGAGGAAGGT 74 GGGTAGGGGTCCTGAGAGGGAGATC 75 Aeromonas
(genus) GAGTGCCTTCGGGAATCAGAACAC 76 CTGCAAGCTAGCGATAGTGAGCGA 77
Bacteroides (genus) CGATGGATAGGGGTTCTGAGAGGAA 78
TGCGGCTCAACCGTAAAATTGCAGT 79 TGTGGCTCAACCATAGAATTGCCGT 80
Peptococcus niger GTACCTGTAAGAAAGACGGCCTTCGT 81
CTGCCGAGTGATGTAATGTCACTTTTC 82 TCGGAGGTTTCAAGACCGTCGG 83
Clostridium (genus) ACCAAAGGAGCAATCCGCTATGAGATG 84
ATCAAAGGTGAGCCAGTACAGGATGG 85 ATTAAAGGAGTAATCCGCTATGAGATGGACC 86
Propionibacterium acnes GGGCTAATACCGGATAGGAGCTCCTG 87
AAGCGTGAGTGACGGTAATGGGTAAA 88 ATCGCGTCGGAAGTGTAATCTTGGG 89
Campylobacter (genus) TGGAGCAAATCTATAAAATATGTCCCAGT 90
ACAGTGGAATCAGCGACTGGGG 91 Aeromonas hydrophila
GCCTAATACGTATCAACTGTGACGTTAC 92 GCCTAATACGTGTCAACTGTGACGTTAC 93
Propionibacterium (genus) GCTTTCGATACGGGTTGACTTGAGGAA 94
Those probes are capable of specifically detecting certain
bacterial species (or genera) shown in the left column in the table
just as one specific to Prevotella denticola of Example 1.
Further, those probes are designed such that they have the same Tm
value as that of a target, the same reactivity with a non-target
sequence, and the like so that the nucleic acid of the bacterial
species of interest can be specifically detected under the same
reaction conditions.
For the respective probes, probe solutions were prepared in a
manner similar to the stage 4-3 of Example 1. Subsequently, the
inkjet printer used in the stage 4-4 of Example 1 was employed to
discharge each of the probe solution on the same substrate to form
a plurality of DNA chips having spots of the respective probes
being arranged at a pitch of about 120 .mu.m.
One of the DNA chips was used for hybridization with the nucleic
acid extracted from Prevotella denticola in a manner similar to the
stage 6 of Example 1. As a result, the spot of the probe which
specifically detected Prevotella denticola showed almost the equal
fluorescence intensity as that of Example 1. In contrast, the spots
of other probes showed extremely low fluorescence intensity.
Further, other prepared DNA chips were used for hybridization with
the bacteria listed in Table 8 except of Prevotella denticola. As a
result, the spot of Prevotella denticola showed extremely low
fluorescence intensity, while the spot of the probe for the
bacterial species of interest showed extremely high fluorescence
intensity. Therefore, the DNA chip prepared in the present example
was confirmed that it was able to simultaneously determine 15
bacterial species and 7 genera listed in Table 8 in addition to
Prevotella denticola. By simultaneously using probes for a target
species and the corresponding genus (for example, Propionibacterium
(genus) and Propionibactrium acnes), highly accurate identification
of the target species or simultaneous identification of a plurality
of target species of the same genus can be performed
Example 3
Using the DNA chip prepared in Example 2, detection was attempted
when a plurality of bacterial species was present in an
analyte.
A culture medium in which Prevotella denticola and Eggerthella
lenta were cultured was prepared and subjected to the same
treatment as that of Example 1 to react with the DNA chip.
As a result, only the spots of the probes having SEQ ID NOS. 49,
50, 51, 70, 71, and 72 showed high fluorescence intensity, so the
coexistence of those bacteria was able to be simultaneously
confirmed.
The present invention is not limited to the above-mentioned
embodiments and various changes and modifications can be made
within the spirit and scope of the present invention. Therefore to
apprise the public of the scope of the present invention, the
following claims are made.
This application claims the benefit of Japanese Patent Application
No. 2006-306007, filed Nov. 10, 2006, which is hereby incorporated
by reference in its entirety.
SEQUENCE LISTINGS
1
95123DNAArtificialPrimer 1gcggcgtgcc taatacatgc aag
23223DNAartificialPrimer 2gcggcaggcc taacacatgc aag
23323DNAartificialPrimer 3gcggcaggct taacacatgc aag
23423DNAartificialPrimer 4gcggtaggcc taacacatgc aag
23523DNAartificialPrimer 5gcggcgtgct taacacatgc aag
23623DNAartificialPrimer 6gcgggatgcc ttacacatgc aag
23723DNAartificialPrimer 7gcggcatgcc ttacacatgc aag
23823DNAartificialPrimer 8gcggcatgct taacacatgc aag
23923DNAartificialPrimer 9gcggcgtgct taatacatgc aag
231023DNAartificialPrimer 10gcggcaggcc taatacatgc aag
231123DNAartificialPrimer 11gcgggatgct ttacacatgc aag
231223DNAartificialPrimer 12gcggcgtgcc taacacatgc aag
231323DNAartificialPrimer 13gcggcgtgca taacacatgc aag
231423DNAartificialPrimer 14gcggcatgcc taacacatgc aag
231523DNAartificialPrimer 15gcggcgcgcc taacacatgc aag
231623DNAartificialPrimer 16gcggcgcgct taacacatgc aag
231723DNAartificialPrimer 17gcgtcatgcc taacacatgc aag
231823DNAartificialPrimer 18gcgataggct taacacatgc aag
231923DNAartificialPrimer 19gcgacaggct taacacatgc aag
232023DNAartificialPrimer 20gctacaggct taacacatgc aag
232123DNAartificialPrimer 21acagaatgct taacacatgc aag
232222DNAartificialPrimer 22atccagccgc accttccgat ac
222322DNAartificialPrimer 23atccaaccgc aggttcccct ac
222422DNAartificialPrimer 24atccagccgc aggttcccct ac
222522DNAartificialPrimer 25atccagccgc accttccggt ac
222622DNAartificialPrimer 26atccagcgcc aggttcccct ag
222722DNAartificialPrimer 27atccagccgc aggttctcct ac
222822DNAartificialPrimer 28atccagccgc acgttcccgt ac
222923DNAartificialPrimer 29taccttgtta cgacttcacc cca
233023DNAartificialPrimer 30taccttgtta cgacttcgtc cca
233123DNAartificialPrimer 31taccttgtta cgacttagtc cca
233223DNAartificialPrimer 32taccttgtta cgacttagcc cca
233323DNAartificialPrimer 33taccttgtta cgacttagtc cta
233423DNAartificialPrimer 34taccttgtta cgacttagcc cta
233528DNAArtificialprobe 35tcatcttgag gtatggaagg gaaagtgg
283628DNAartificialprobe 36gtgttaggtg tctggaataa tctgggtg
283725DNAartificialprobe 37accaagtctt gacatattac ggcgg
253826DNAartificialprobe 38aaggattccg gtaaaggatg gggatg
263926DNAartificialprobe 39tggaaacatg tcagtgagca atcacc
264027DNAartificialprobe 40aagaatttcg gttatcgatg gggatgc
274129DNAartificialprobe 41aagttttcca cgtgtggaat tttgtatgt
294224DNAartificialprobe 42aaggcagcta cctggtgaca ggat
244330DNAartificialprobe 43aatatcaaag gtgagccagt acaggatgga
304428DNAartificialprobe 44ccgtagtaag ctcttgaaac tgggagac
284528DNAartificialprobe 45tcccaatgac atctccttaa tcggagag
284627DNAartificialprobe 46aaccaaagga gcaatccgct atgagat
274725DNAartificialprobe 47gagcgtaggc ggatgattaa gtggg
254829DNAartificialprobe 48cccttgcatt actcttaatc gaggaaatc
294922DNAartificialprobe 49ggaaagccca gacggcaagg ga
225026DNAartificialprobe 50cctctcaagc gggatctcta atccga
265123DNAartificialprobe 51tgccccatgt tgccagcatt agg
235228DNAartificialprobe 52ttttcgcatg gaggaatcat gaaagcta
285321DNAartificialprobe 53agatgcgccg gtgccctttc g
215426DNAartificialprobe 54agtcgggaag aagtcagtga cggtac
265524DNAartificialprobe 55gagtacgtgc gcaagcatga aact
245624DNAartificialprobe 56gaagactgcc cgcaagggtt gtaa
245725DNAartificialprobe 57gtgtactgga ggtacgtgga acgtg
255824DNAartificialprobe 58gcatgaggct gagaggtctc ttcc
245928DNAartificialprobe 59ttatagctgt aagataggca tgcgtccc
286026DNAartificialprobe 60aacgggcgat acgagtattg cattga
266125DNAartificialprobe 61atataccgtc aagcttccac agcga
256225DNAartificialprobe 62tttgaaaggc gcttttgcgt cactg
256330DNAartificialprobe 63caaggatgag agtagaacgt tcatcccttg
306430DNAartificialprobe 64caaggatgag agtagaatgt tcatcccttg
306527DNAartificialprobe 65cctgggaatg gcatctaaga ctggtca
276628DNAartificialprobe 66gaggaaggcg ttgatgctaa tatcatca
286724DNAartificialprobe 67gagcaaagca ggggaacttc ggtc
246824DNAartificialprobe 68gttggggcct ttgaggcttt agtg
246926DNAartificialprobe 69tgggagagga tggtagaatt ccaggt
267026DNAartificialprobe 70tcgatgacgg catcagattc gaagca
267124DNAartificialprobe 71aatgtaggcg cccaacgtct gact
247224DNAartificialprobe 72atgttgaggt ccttcgggac tcct
247331DNAartificialprobe 73ggaagtaact agaatatgta gtgtagcggt g
317424DNAartificialprobe 74gccagtgcaa actgtgagga aggt
247525DNAartificialprobe 75gggtaggggt cctgagaggg agatc
257624DNAartificialprobe 76gagtgccttc gggaatcaga acac
247724DNAartificialprobe 77ctgcaagcta gcgatagtga gcga
247825DNAartificialprobe 78cgatggatag gggttctgag aggaa
257925DNAartificialprobe 79tgcggctcaa ccgtaaaatt gcagt
258025DNAartificialprobe 80tgtggctcaa ccatagaatt gccgt
258126DNAartificialprobe 81gtacctgtaa gaaagacggc cttcgt
268227DNAartificialprobe 82ctgccgagtg atgtaatgtc acttttc
278322DNAartificialprobe 83tcggaggttt caagaccgtc gg
228427DNAartificialprobe 84accaaaggag caatccgcta tgagatg
278526DNAartificialprobe 85atcaaaggtg agccagtaca ggatgg
268631DNAartificialprobe 86attaaaggag taatccgcta tgagatggac c
318726DNAartificialprobe 87gggctaatac cggataggag ctcctg
268826DNAartificialprobe 88aagcgtgagt gacggtaatg ggtaaa
268925DNAartificialprobe 89atcgcgtcgg aagtgtaatc ttggg
259029DNAartificialprobe 90tggagcaaat ctataaaata tgtcccagt
299122DNAartificialprobe 91acagtggaat cagcgactgg gg
229228DNAartificialprobe 92gcctaatacg tatcaactgt gacgttac
289328DNAartificialprobe 93gcctaatacg tgtcaactgt gacgttac
289427DNAartificialprobe 94gctttcgata cgggttgact tgaggaa
27951493DNAPrevotella denticola 95ctcaggatga acgctggcta caggcttaac
acatgcaagt cgaggggaaa cggcattgag 60tgcttgcact gaatggacgt cgaccggcgc
acgggtgagt aacgcgtatc caaccttccc 120gttactgcgg gataacctgc
cgaaaggcag actaataccg catgttcttc gatgacggca 180tcagattcga
agcaaagaty crtcggtaac ggagggggat gcgtctgatt agctagttgg
240cggggcgacg gcccaccaag gckacgatca gtaggggttc tgagaggaag
gtcccccaca 300ttggaactga gacacggtcc aaactcctac gggaggcagc
agtgaggaat attggtcaat 360gggcggaagc ctgaaccagc caagtagcgt
gcaggakgac ggccctacgg gttgtaaact 420gcttttatgc ggggataaag
tgagggacgy gtcccttttt gcaggtaccg catgaataag 480gaccggctaa
ttccgtgcca gcagccgcgg taatacggaa ggtccgggcg ttatccggat
540ttattgggtt taaagggagc gtaggccggg gattaagtgt gttgtgaaat
gtaggcgccc 600aacgtctgac ttgcagcgca tactggttcc cttgagtacg
cgcaacgccg gcggaattcg 660tcgtgtagcg gtgaaatgct tagatatgac
gaagaacccc gattgcgaag gcagccggcg 720ggagcgcaac tgacgctgaa
gctcgaaggt gcgggtatcg aacaggatta gataccctgg 780tagtccgcac
ggtaaacgat ggatgcccgc tgtcggcgcc ttgcgccggc ggccaagcga
840aagcgttaag catcccacct ggggagtacg ccggcaacgg tgaaactcaa
aggaattgac 900gggggcccgc acaagcggag gaacatgtgg tttaattcga
tgatacgcga ggaaccttac 960ccgggcttga atygcaggag aacgatacag
agatgttgag gtccttcggg actcctgcga 1020aggtgctgca tggttgtcgt
cagctcgtgc cgtgaggtgt cggctyaagt gccataacga 1080gcgcaacccc
tctccccagt tgccatcggg tgatgccggg cactccgggg acactgccgc
1140cgcaaggtgc gaggaaggcg gggaygacgt caaatcagca cggcccttac
gtccggggct 1200acacacgtgt tacaatggcc ggcacagagy gcyggtgcgg
ygcgagccgc atcyaatctt 1260gaaaaccggt ctcagttcgg actggggtct
gcaacccgac cccacgaagc tggattcgct 1320agtaatcgcg catcagccac
ggcgcggtga atacgttccc gggccttgta cacaccgccc 1380gtcaagccat
gaaagccggg ggtgcctgaa gtccgtgacc gcgaggatcg gcctagggca
1440aaactggtga ttggggctaa gtcgtaacaa ggtagccgta ccggaaggtg cgg
1493
* * * * *